Quantitative polymerase chain reaction (qPCR) is a rapid molecular method that can be used to detect and quantitate Legionella pneumophila and Legionella pneumophila serogroup 1 in potable or non-potable water samples. This is achieved by amplification of a target gene sequence that is unique to the genome DNA of L. pneumophila or L. pneumophila serogroup 1. The amplified sequence is detected using a fluorescent signal. Approximations of amount of target gene detected can be determined as low, moderate, or high by comparing the amount of fluorescence of the target gene to the amount of fluorescence of a known quantity of DNA (i.e. comparing to a standard curve). Testing can be performed in approximately 2 to 4 days as compared to 7 to 10 days for culture. The procedure follows International Standard Organization’s (ISO) standard 12869 with minor modifications.
The qPCR assay cannot discriminate the genome of live vs dead/impaired L. pneumophila and may also detect L. pneumophila that are not culturable. Because of this, genome copies (genomic units) cannot be directly correlated to CFU.
There are more than 50 Legionella species, however L. pneumophila (specifically L. pneumophila serogroup 1), are responsible for a large majority of illness. Therefore the qPCR test is designed to identify or rule out only the most clinically relevant strains. If requested, or when species identification is required such as in investigations of clinical cases, culture identification and DNA sequencing can be performed for identification of species other than L. pneumophila.
- SPL. M17.01 Quantitative Polymerase Chain Reaction (qPCR) For Detection of L. pneumophila and L. pneumophila, serogroup 1.
- Water samples are concentrated by filtration.
- DNA is extracted using the Mo-Bio PowerWater Kit.
- Taqman qPCR is performed on the extracted DNA with two primer/probe sets: 1) Legionella pneumophila specific for a sequence within the 16s rRNA gene; 2) Legionella pneumophila serogroup 1 specific for a sequence within the LPS gene.
- Amount of L. pneumophila genome present is extrapolated using a standard curve, and reported as not detected, low, moderate, or high level present (serogroup 1 reported as detected or not).
Turn Around Time
Sample Collection and Shipping
- Please see Legionella Sampling and Shipping for sampling guidelines.
- Ship the same day samples are collected for receipt in the laboratory.
- Place bottles in box with insulated liner.
- Place completed Chain of Custody form in plastic document holder and pack in box.
- Ship overnight Monday–Thursday only: Special Pathogens Laboratory, 1401 Forbes Avenue, Suite 401, Pittsburgh, PA 15219.
- qPCR Application Guide – Experimental Overview, Protocol, Troubleshooting. 3rd Edition. 2011-2012. Integrated DNA Technologies.
- Mo-Bio. RapidWater. DNA Isolation Kit Instruction Manual.
- Applied Biosystems. Quantstudio 6 and 7 Flex Real-Time PCR System Software. 2013. Booklets 2 and 5.
- Donohue MJ, O'Connell K, Vesper SJ, Mistry JH, King D, Kostich M, Pfaller S. Widespread molecule detection of Legionella pneumophila Serogroup 1 in cold water taps across the United States. Environ Sci Technol. 2014 Mar 18; 48(6):3145-52.
- ISO Standard 12869: 2012. Water Quality – Detection and quantification of Legionella spp. by concentration and genic amplification by quantitative polymerase chain reaction (qPCR)